BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt

Abstract

Background: Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS.

Methods: The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment.

Results: BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity.

Conclusions: BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.

Figure 1

Anti-proliferative effect of BI-69A11 on colon cancer cells. (A) Dose-dependent growth inhibitory effects of BI-69A11 on colorectal cancer cell lines. HCT116, HT29, HCT15 and SW480 cells were treated with various concentrations of BI-69A11 and incubated for 12, 24 and 48 h, respectively, and MTT assays were performed. Points, average±s.d. of three different experiments each performed in triplicate, P<0.05. (B) The indicated cells were treated with an IC₅₀ dose of BI-69A11 for indicated time (hrs). Cells were stained with calcein AM and ethidium bromide and images were captured. Graphical representations of live and dead cells in the experiment are shown as histograms; *P<0.05, **P<0.01, ***P<0.001 represents level of significance with respect to control. (C) Cell cycle analysis was performed by treating the indicated cells with an IC₅₀ dose of BI-69A11. Cells were exposed to the indicated concentrations of BI-69A11 for 3, 6, 12 and 24 h followed by propidium iodide staining. The data represent the percentage accumulation of cells in each phase and are representative of three independent experiments.

Figure 2

BI-69A11 induces apoptosis of colon cancer cells. (A) Characteristic apoptotic cells were detected in HCT116 and HT29 cell lines treated with BI-69A11 for 12 and 24 h by staining with DAPI. Photographs were taken under 20 × magnifications using a confocal microscope. (B) TUNEL assays were performed as per the manufacturers' protocol on HCT116 and HT29 cells by treating cells for the indicated times with BI-69A11. The apoptotic cells with DNA fragmentation are stained positively as green nuclei and live cells with intact nuclei are stained as red nuclei. Both photographs were taken at 20 × magnification and are representative of three separate experiments. (C) Western blotting of HCT116 and HT29 cells treated with BI-69A11 for the indicated times. Representative figures of three independent experiments. (D) Apoptosis was determined by flow-cytometric detection of Annexin V-FITC-positive cells treated for the indicated hours with BI-69A11. Representative histograms from three independent experiments are shown. The relative number of cells in each quadrant is given in per cent. **P<0.01, ***P<0.001 represents level of significance with respect to control.

Figure 3

BI-69A11 inhibits Akt phosphorylation and phosphorylation of downstream targets of Akt in colon cancer cells. (A) and (E) HCT116 and HT29 colon cancer cells were untreated or treated with IGF (100 ng ml⁻¹) and/or BI-69A11 (5 μM) and equal amounts of protein cell lysates were analysed by Western blotting. (B) Representative densitometric analysis of Western blots as described in panel A and, the degree of inhibition by BI-69A11 in both Ser473 and Thr308 residues, with respect to total Akt was determined. Data are means±s.e. of three random experiments; *P<0.05, **P<0.01, ***P<0.001 represent level of significance with respect to control. (C) HCT116 and HT29 cells were treated with or without 5 μM of BI-69A11 or GSK690693 for 1 h and cell lysates were analysed for phospho-GSK-3β for Akt kinase activity and total GSK-3β. (D) The expression of Akt kinase activity was determined by densitometry and is shown in the graph. Data are means±s.e. of three random experiments; *P<0.05, **P<0.01, ***P<0.001 represents level of significance with respect to control.

Figure 4

BI-69A11 suppresses the migration and invasion of colon cancer cells. (A) Microscopic observation of the growth of HCT116 and HT29 cells after sub-lethal dose of BI-69A11 exposure for the indicated times (h). A ‘wound' was created with a pipette tip before treatment with 0.5 μM BI-69A11 containing culture medium. The data are representative of three independent experiments. Photographs were taken at 4 × magnification. (B) Quantification of wound-healing results. Data are means±s.e. of three random widths along the wound with time. *P<0.05, **P<0.01, ***P<0.001 represent level of significance with respect to control. (C) Data represent the average percentage of cells (±s.d.) invading the Boyden chamber inserts coated with Matrigel from three independent experiments, each performed in triplicate; *** represents P<0.0001. For this experiment HT29 cells were treated with 0.5 μM of BI-69A11.

Figure 5

Combinatorial antitumour effects of Ad.5/3-mda-7 and BI-69A11 on colon cancer cells. (A) HT29 and HCT116 cells were infected with 25 pfu per cell of Ad.5/3-vec or Ad.5/3-mda-7 for 6 h and then treated with the indicated concentrations of BI-69A11 for 48 h and cell viability was monitored by MTT assay±s.d. (n=3); P<0.01 for the BI-69A11– and Ad.5/3-mda-7-treated groups. (B) HT29 and HCT116 cells were infected with Ad.5/3-vec or Ad.5/3-mda-7 for 6 h and then exposed to the IC₅₀ of BI-69A11 for 48 h and cells were lysed and Western blotting was performed with the indicated antibodies. Each experiment was performed in triplicate.

Figure 6

Combinatorial effect of Ad.5/3-mda-7 and BI-69A11 inhibits colon cancer cell migration, invasion and angiogenesis. (A) CAM assay was performed by treating fertilised (day 0) eggs with Ad.5/3-vec, VEGF, BI-69A11, Ad.5/3-mda-7, Ad-5/3-mda-7 and BI-69A11 or with Ad.5/3-mda-7, BI-69A11 and VEGF. Photomicrographs were taken at 20 × magnification. (B) Graphical representations of quantification of CAM assays as shown in panel A; (C) HUVEC cells were seeded in 96-well plates and treated with Ad.5/3-vec, VEGF as a positive control, BI-69A11, Ad.5/3-mda-7 and BI-69A11 or Ad.5/3-mda-7, VEGF, BI-69A11 and Ad.5/3-mda-7. Photomicrographs were taken at 40 × magnification. (D) Graphical representations of quantification of HUVEC assays; *P<0.05, **P<0.01, ***P<0.001 represent level of significance with respect to Ad.5/3-vec in (B) and (D) by unpaired t-test.

Figure 7

Effects of Ad.5/3-vec, Ad.5/3-mda-7 and BI-69A11 on invasion of HT29 colon carcinoma cells. (A) Photomicrographs of HT29 cell invasion assays following BI-69A11 and Ad.5/3-mda-7 infection alone and in combination. HT29 cells were treated with 0.5 μM BI-96A11 and/or Ad.5/3-mda-7 (25 pfu per cell) for 48 h. Photographs were taken at 10 × magnification. (B) Data represent the average percentage of cells (±s.d.) invading the Boyden chamber inserts coated with Matrigel of three different experiments, each performed in triplicate; **P<0.01 and ***P<0.001 represent level of significance with respect to Ad.5/3-vec.

Figure 8

The combination of Ad.5/3-mda-7 and BI-69A11 inhibits the growth of human colon cancer xenografts in nude mice. (A) Measurement of HT29 xenograft tumour volumes at different time points. Data presented as mean±s.d. (n=5), P<0.05, when compared with the Ad.5/3-vec-treated group. (B) Representative images and measurements of the tumour weights at the end of the study. Columns, mean±s.d. (n=5). (C) Immunohistochemistry of BI-69A11- and Ad.5/3-mda-7-treated HT29 colon cancer xenografts. Paraffin-embedded sections of HT29 bearing tumours in nude mice were processed and IHC was done after staining with Akt, p-Akt, ribosomal-S6 protein and p-ribosomal-S6 protein to study the Akt pathway. Staining with Ki-67 and CD31 was used to monitor the anti-proliferative effect of single- and combination-treated tumours. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling) assays were performed to study apoptosis. Pictures were taken at a magnification 20 × .